Clostridium difficile is the leading cause of hospital-acquired diarrhea in developed countries. This spore-forming anaerobic bacterium contaminates hospital environments and infects patients undergoing antibiotic therapy within health care facilities. Despite these problems, historically, treatment with antibiotics such as metronidazole and vancomycin has been an effective means of treating this disease. Yet, disturbing trends of increased morbidity and mortality, as well relapse of C. difficile infected patients have become apparent over the past decade. These trends correlate with an increase in the number of patients infected by a hypervirulent strain of C. difficile. This C. difficile strain, often referred to as the BI/NAP1/027 strain, has now been found in a majority of states in the US and is prominent both in Europe and Canada. To date, many factors such as antibiotic resistance, sporulation ability, and toxin production have been proposed to contribute to the difference in virulence of historical and hypervirulent C. difficile. Yet, the relevance of these factors is still greatly debated, leaving us with a poor understanding into how this emergent strain correlates with increased mortality.
C. difficile produces two large clostridial toxins, TcdA and TcdB, which cause extensive tissue damage and ultimately lead to human disease. Our work has focused on understanding how variations in the toxins produced by historical and epidemic strains change the extent of C. difficile virulence. Of particular interest are the differences in the sequence and activities of TcdB, which has been implicated as a critical C. difficile virulence factor.
TcdB is a single chain polypeptide toxin where the glucosyltransferase domain is located at the N-terminus (GTD: 1-543), followed by an autoprocessing site between amino acid 543 and 544 which is subject to intramolecular cleavage by the cysteine protease domain (CPD: 544-807), a hydrophobic transmembrane domain (TMD: 956-1128), and a putative receptor binding domain at the C-terminus (CTD: 1651-2366). The gene encoding TcdB is located within a pathogenecity locus on the chromosome of C. difficile along with genes encoding TcdA (enterotoxin), TcdE, and regulators of toxin gene expression (TcdC and TcdR). While the sequence of TcdA, TcdE, TcdR, and TcdC are almost identical between historical and hypervirulent strains, TcdB is more variable (96% similarity, 92% identity). TcdB from a hypervirulent strain (TcdBHV) has been found to be more potent on cultured cells than TcdB from a historical strain (TcdBHIST). In line with this we also found that TcdBHV caused more extensive and broader tissue pathologies in a zebrafish embryo model. As a possible underlying mechanism for these differences in activity, it has been found previously that TcdBHV is translocated into cells more rapidly and is autoprocessed more efficiently than TcdBHIST.
Interestingly, the greatest sequence variation between the two forms of TcdB is found in the C-terminal domain (CTD), which we define as the region of the toxin between amino acid 1651 and the terminal residue at position 2366. There is an overall 88% sequence identity between TcdBHV1651-2366 and TcdBHIST 1651-2366. The CTD of TcdB encodes combined repetitive oligopeptides (CROPs), which are thought to be responsible for the recognition of glycans on target cells, and as such the CTD is often referred to as the receptor binding domain. However, the role of the CTD as the receptor binding domain is still very much debated as no receptor has been identified, and studies in TcdA have shown that this region contributes to, but is not required for cellular uptake of the toxin. The CTD is also antigenic and known to contain neutralizing epitopes. Yet, whether sequence differences in the CTD of TcdBHV and TcdBHIST alter the tropism or antigenic profiles of these two forms of the toxin is not known.
Upon examining the differences in the lethality and in vivo pathologies of TcdBHV and TcdBHIST, the data indicates that TcdBHV exhibits a lethal dose substantially lower than TcdBHIST. Additionally, while both toxins caused pronounced hemorrhaging in major organs, TcdBHV causes brain pathologies in vivo as well as an increased cytotoxicity on brain microvascular cells in vitro.
The use of C. difficile toxin as a vaccine has been tried by others, but has had limited success. Although the use of the vaccine results in the production of antibodies, this does not always correlate with protection against future C. difficile infection. Therefore, there is a need for a C. difficile vaccine that is capable of conferring protection against future infection.